The U.S. dairy industry is largely comprised of farmer-owned dairy cooperatives and proprietary companies. The cooperatives tend to specialize in fresh milk and butter where proprietary companies control branded cheeses and cultured products. Through the use of technology, the dairy industry has increased milk production over the past 10 years. The number of dairy operations has declined while per animal efficiency has increased. Due to intense milking schedules and loss of nutrients involved with the milking process, health problems for dairy cows have been compounded. This ultimately costs dairy producers, which in turn costs dairy consumers. Early detection of health problems in dairy cattle could prevent death loss and financial burdens on the dairy industry.
A common problem in dairy cattle is hypocalcemia or “milk fever.” Milk fever in dairy cows results from an acute deficiency of blood calcium which typically occurs in the time immediately before calving. While the problem exists in all cows, it is more acute in dairy cattle. It has been reported that the average incidence of milk fever is about 6% in U.S. dairy herds, although it may be much higher on individual farms (Sanchez et al., 1992). Calving causes a high volume of milk production, and with it, a high demand for calcium from the cow's body. If the body is unable to respond quickly to this demand, the cow develops hypocalcemia, or an abnormally low blood-calcium level. Hypocalcemic cows will begin trembling and, as calcium level continues to plummet, will no longer be able to stand. Left untreated, most affected cows will die. Shortly before calving, large amounts of calcium are removed from the blood and are utilized in the mammary gland to be part of the colostrum, defined as milk that is secreted for a few days after parturition and characterized by high protein and antibody content. Calcium in the colostrum may be 8 to 10 times greater than that in the blood supply. The rapid drop and the decreased mass of the calcium pool prior to parturition, and the failure of calcium absorption to increase fast enough after the onset of lactation, can predispose animals to hypocalcemia. A cow afflicted with acute milk fever may remain alert, eat, and milk, but is not able to stand. The cow may become a “creeping downer” that has flexed pasterns and posterior paralysis. The condition can lead to rupture of the large muscle or group of muscles in one or both of the hind legs. In this downer condition, the cow is also susceptible to fracture or dislocation of a hind joint when the cow initially goes down or struggles to rise, as well as possible damage to the udder which can lead to mastitis.
Milk fever can also result in lost milk production, poor reproductive performance, increased culling rates, additional labor costs to treat afflicted animals, and animal death. Subacute milk fever causes cows to be easily excited, with symptoms of muscle twitching and tremors. The onset of milk fever causes notable changes in the blood parameters, especially blood calcium. Normal blood calcium (total calcium levels) in an adult dairy cow generally ranges from 8-10 mg per dl. In stage one hypocalcemic cows, blood calcium levels range from 5.5 to 7.5 mg per dl. Stage two hypocalcemia is characterized by blood calcium levels of 3.5 to 6.5 mg per dl. In stage three, calcium levels may drop as low as 2.0 mg per dl. The drop in blood calcium levels is usually accompanied by a drop in phosphorus concentrations and an increase in magnesium concentration (Smith, 1996).
Traditional tests for calcium levels involve testing blood serum. This requires taking a blood sample from the “patient,” and running that sample through a standard test to determine the complete calcium level in the sample. Essentially all of the calcium found in blood is present in the plasma as two distinct forms: nondiffusible protein-bound calcium (bound to albumin, for instance) and the diffusible free calcium fraction. Of the two forms, the nondiffusible protein-bound fraction constitutes roughly 40% to 50% of the total extracellular calcium, while the diffusible free fraction can be further subdivided into ionized calcium (which is the physiologically active form) and complexed calcium (which is found bound to bicarbonate, citrate, phosphate, and sulfate).
The usual calcium level test determines the total calcium levels (both diffusible and non-diffusible calcium), as testing for ionic calcium is a more burdensome test. The total calcium test is a good reflection of the amount of free calcium involved in metabolism since the balance between free and bound is usually stable and predictable. Methods to test a blood serum sample for total calcium include atomic absorption spectrophotometry analysis, oxalate precipitation followed by titration, fluorometric, titrimetric, flame photometry and photometric determination (as exemplified in U.S. Pat. No. 4,871,678, or descried in Gindler E M, King J D. Rapid Colorimetric Determination of Calcium in Biologic Fluids with Methylthymol Blue. Am J Clin Path 1972; 58:376-382. 22). Photometric determination involves the use of dye complexes (such as methylxyenol blue, arsenazo III or o-cresolphthalein complexone) that reacts with calcium in a particular environment (acidic, buffered, etc) yielding a colored calcium-dye complex. The resulting increase in absorbance caused by the formation of the complex is bichromatically measured (such as by spectrophotometric means) and correlated to the calcium concentration of the sample. See, in general, Burtis and Ashwood, Tietz Textbook of Clinical Chemistry, 2nd Ed, W. B. Saunders 1994, chap 36, hereby incorporated by reference, which describes several of the methods above. Due to the importance of calcium in biological systems, the attention paid to determining calcium levels is reflected in a number of patents addressing the issue, such as U.S. Pat. No. 5,618,684 (Method of determination of calcium); U.S. Pat. No. 5,482,866 (Method for quantization of calcium and magnesium and the novel reagent compositions); U.S. Pat. No. 5,262,330 (Colorimetric methods and reagents for the assay of calcium in a test); U.S. Pat. No. 5,057,435 (Reagent and methods for calcium determination); U.S. Pat. No. 4,795,712 (Calcium complexing dyes and their use in analytical compositions, elements and methods).
While serum calcium levels will reveal calcium levels which are used to diagnose hypocalcemia, treatment is usually initiated based on clinical signs only (i.e., often at advanced stages of the condition) because of the slow return of laboratory blood tests and the rapid nature of this illness. Affected cows have an excellent prognosis if the condition is diagnosed and treated early and properly. Late diagnosis and delayed treatment may result in a comatose animal with a much poorer prognosis. If treatment is not successful, death of the animal results. Animal survival is favorable with early treatment by intravenous calcium supplementation. However, repeated treatments with intravenous calcium can suppress homeostatic mechanisms for increasing serum calcium (Smith, 1996), suppressing the animal's normal mechanism for regulating serum calcium thereby compounding the problem. Consequently, the need for repeated treatment should be based on laboratory verification of low serum calcium levels. Treatment costs for milk fever are estimated to be about $334 per head (Smith, 2002).
Treatment for milk fever has been estimated to cost the dairy industry $167 million annually (excluding death loss). Other than the current blood serum tests, the dairy industry has no proven technique for accurately measuring calcium levels. Blood serum tests, if undertaken at all, are typically “too little, too late” for the animal and the dairy. By the time symptoms are present, milk production has already been significantly affected and the animal is in danger. The ability to monitor bovine blood calcium concentrations in real-time without any disease indicator would be beneficial in possibly preventing, diagnosing, and/or treating hypocalcemia.
The basic problem is that traditional tests for calcium levels in cows require a blood sample, and as the sample must be sent to a lab for analysis, traditional methods entail a delay in results, coupled with a costly test. Proper handling of the blood sample is problematic, requiring cooling and centrifugation to produce blood plasma or serum. In addition, the collection of blood specimens from the jugular vein, the anterior mammary (milk) vein, or the coccygeal grove at the top of the tail head would require frequent animal venipuncture. This could result in undesirable animal behavior, increased animal stress, and reduced milk production. It would be advantageous to avoid taking a blood sample, and further to have an efficient and easily administered means for monitoring calcium concentrations in the animal, preferably at “cow-side” before symptoms develop or advance so the appropriate measures, such as administration of subcutaneous, intraperitoneal, oral, or intravenous calcium products, can be taken to avoid or early-treat milk fever. An alternative method of monitoring blood calcium levels may be possible, provided that there is a correlation between blood calcium concentrations and calcium concentrations in other body fluids that may be readily accessed without venipuncture.